IDENTIFICATION OF A KILLER CELL-SPECIFIC REGULATORY ELEMENT OF THE MOUSE PERFORIN GENE - AN ETS-BINDING SITE-HOMOLOGOUS MOTIF THAT INTERACTS WITH ETS-RELATED PROTEINS

The gene encoding the cytolytic protein perforin is selectively expres sed by activated killer lymphocytes. To understand the mechanisms unde rlying the cell-type-specific expression of this gene, we have charact erized the regulatory functions and the DNA-protein interactions of th e 5'-flanking region of the mouse perforin gene (Pfp). A region extend ing from residues +62 through -141, which possesses the essential prom oter activity, and regions further upstream, which are able to either enhance or suppress gene expression, were identified. The region betwe en residues -411 and -566 was chosen for further characterization, sin ce it contains an enhancer-like activity. We have identified a 32-mer sequence. (residues -491 to -522) which appeared to be capable of enha ncing gene expression in a killer cell-specific manner. Within this se gment, a 9-mer motif (5'-ACAGGAAGT-3', residues -505 to -497; designat ed NF-P motif), which is highly homologous to the Ets proto-oncoprotei n-binding site, was found to interact with two proteins, NF-P1 and NF- P2. NF-P2 appears to be induced by reagents known to up-regulate the p erforin message level and is present exclusively in killer cells. Elec trophoretic mobility shift assay and UV cross-linking experiments reve aled that NF-P1 and NF-P2 may possess common DNA-binding subunits. How ever, the larger native molecular mass of NF-P1 suggests that NF-P1 co ntains an additional non-DNA-binding subunit(s). In view of the homolo gy between the NF-P motif and other Ets proto-oncoprotein-binding site s, it is postulated that NF-P1 and NF-P2 belong to the Ets protein fam ily. Results obtained from the binding competition assay, nevertheless , suggest that NF-P1 and NF-P2 are related to but distinct from Ets pr oteins, e.g., Ets-1, Ets-2, and NF-AT/Elf-1, known to be expressed in T cells.